Rapamycin derivative with antimicrobial, anticancer and immunomodulation activity

ABSTRACT

A compound of formula (I) is obtainable by the cultivation of a genus Streptomyces, such as Streptomyces NCIMB 40515. in the presence of rapamycin, and recovery thereof from the culture medium. The compound, and pharmaceutical compositions containing it, is useful as an immunomodulatory agent as an anticancer agent, or in the treatment of microbial infections in animals including humans. ##STR1##

This application is a 371 of PCT/GB 94/01600, filed 25 Jul. 1994, whichclaims priority of British Application 9315914.3 filed 31, Jul. 1993.

This application is a 371 of PCT/GB 94/01600, filed 25 Jul. 1994, whichclaims priority of British Application 9315914.3 filed 31, Jul. 1993.

The present invention relates to a novel compound and derivatives toprocesses for their production, to pharmaceutical formulationscontaining them, to their use in medical therapy, particularly in thetreatment of microbial infections, and also to their use as immunomodulatory agents.

Rapamycin a known compound and was first isolated as an extract of thefungus Streptomyces hygroscopicus and reported to have antifungalactivity (British Patent 1436447). Subsequently rapamycin has beenimplicated as an immunosuppressant (Martel R. R. et al Can. J. Physiol.Pharmacol, 55, 48-51, 1977).

A large number of microorganisms have been found to produce a variety ofcompounds which have subsequently been isolated and have been shown topossess useful therapeutic properties. Novel compounds have also beenobtained by the incubation or cultivation of a microorganism in thepresence of known compounds. One such new compound is42-O-demethylrapamycin. This novel compound has been found to haveuseful antimicrobial and anticancer and immunomodulation activity.

Accordingly the present invention provides 42-O-demethylrapamycin andderivatives thereof.

The invention in a second aspect, further provides a process for theproduction of 42-O-demethylrapamycin which comprises contacting amicoorganism with rapamycin and subsequently isolating42-O-demethylrapamycin or derivatives thereof from the incubation.

42-O-demethylrapamycin is believed to have the structure shown informula (I): ##STR2##

This compound is referred to herein as 42-O-demethylrapamycin accordingto the numbering system of J. Findlay et al., Can. J. Chem. (1980) 58,579. However according to the more recent numbering system of J. McAlpine et al., J. Antibiotics (1991) 44. 688 this would be known as39-O-demethylrapamycin.

Following the numbering system according to Chemical Abstracts (11thCumulative Index 1982-86 page 60719CS), the compound of the presentinvention would be called 41-O-demethylrapamycin.

The compound in formula (I) has the following characteristics:

i) it has an apparent molecular weight of 899 by fast atom bombardment(FAB) mass spectroscopy;

ii) it is obtainable by the cultivation of a microorganism from thegenus Streptomyces, in the presence of rapamycin and the recovery of42-O-demethylrapamycin or a derivative thereof from the culture medium;

iii) ¹³ CNMR spectroscopy reveals 50 carbons in the molecule;

iv) it shows antifungal activity;

v) it shows immunomodulatory activity.

The term `cultivation` (and derivatives of that term) as used hereinmeans the deliberate aerobic growth of an organism in the presence ofassimilable sources of carbon, nitrogen, sulphur and mineral salts. Suchaerobic growth may take place in a solid or semi-solid nutritive medium,or in a liquid medium in which the nutrients are dissolved or suspended.The cultivation may take place on an aerobic surface or by submergedculture. The nutritive medium may be composed of complex nutrients ormay be chemically defined.

It has been found that suitable microorganisms for use in the processaccording to the invention include bacterial strains belonging to thegenus Streptomyces which are capable of elaborating42-O-demethylrapamycin. It has further been found that an example ofsuch a strain is sp. NCIMB 40515, which has been isolated from natureand also mutants thereof.

The term `mutant` as used herein includes any mutant strain which arisesspontaneously or through the effect of an external agent whether thatagent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains including those outlined by H. I. Adler in`Techniques for the Development of Microorganisms` in `Radiation andRadioisotopes for Industrial Microorganisms`, Proceedings of aSymposium, Vienna, 1973, page 241, International Atomic EnergyAuthority, and these include:

(i) Ionizing radiation (e.g. X-rays and γ-rays), u.v. light, u.v. lightplus a photosensitizing agents (e.g. 8-methoxypsoralen), nitrous acid,hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil),acridines, alkylating agents (e.g. mustard gas, ethyl-methanesulphonate), hydrogen phenols, formaldehyde, heat, and

(ii) Genetic techniques, including, for example, recombination,transformation, transduction, lysogenisation, lysogenic conversion,protoplast fusion and selective techniques for spontaneous mutants.

Using the methods of Becker B. Lechevalier M. P., Gordon R. E.,Lechevalier H. A., 1964, Appl. Microbiol. 12, 421-423 and Williams S.T., Goodfellow M, Wellington E. M. H., Vickers J. C., Alderson. G.,Sneath P. H. A., Sackin M. J., and Mortimer M. 1983 J. Gen. Microbiol.129, 1815-1830, Sp. NCIMB 40515 has been identified as a previouslyunreported, atypical, strain of Streptomyces and therefore also forms apart of the present invention, particularly in biologically pure form.It has been deposited at the National Collections of Industrial andMarine Bacteria Ltd. (N.C.I.M.B), Aberdeen, Scotland under number NCIMB40515 on 7th Jul. 1992.

The medium for cultivating sp. NCIMB 40515 suitably contains sources ofassimilable carbon and assimilable nitrogen together with inorganicsalts. Suitable sources of nitrogen include yeast extract, soyabeanflour, meat extract, cottonseed, flour, malt, distillers dried solubles,amino acids, protein hydrolysates and ammonium and nitrate nitrogen.Suitable carbon sources include glucose, lactose, maltose, starch andglycerol. Suitably the culture medium also includes alkali metal ions(for example, sodium) halogen ions (for example, chloride), and alkalineearth metal ions (for example calcium and magnesium), as well as traceelements such as iron and cobalt.

The cultivation may suitably be effected at a temperature of about 20°to 35° C., advantageously 20° to 30° C., and the culture may suitably becontacted with rapamycin for up to 7 days, advantageously about 3 to 5days, in order to give an optimum yield of the desired product afterisolation for example as described below.

The desired product or a derivative thereof may be isolated from theculture medium and worked up and purified using conventional techniquesfor such compounds. All such isolation and purification procedures mayconveniently be effected at cool to ambient temperature, for example ata temperature within the range of from 4° to 40° C., conveniently from20° to 35° C.

The desired compound may readily be identified in a routine manner bytesting for biological activity and/or by monitoring the h.p.l.c.retention time.

Suitably, the separation procedure may include a high-performance liquidchromotography step, preferably as the last step. Elution may beeffected using aqueous methanol.

42-O-demethylrapamycin and its derivatives may be crystalline ornon-crystalline and, if crystalline, may optionally be hydrated orsolvated.

The derivatives are preferably pharmaceutically acceptable derivatives.Derivatives may include salts, with pharmaceutically acceptable counterions.

The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitable atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, preferably at least 95% pure, especially at least 98% pure,all percentages being calculated as weight/weight. An impure or lesspure form of a compound according to the invention may, for example, beused in the preparation of a more pure form of the same compound or of arelated compound (for example a corresponding derivative) suitable forpharmaceutical use.

42-O-demethylrapamycin and its pharmaceutically acceptable derivativeshave antifungal activity and are useful for the prophylactic andtherapeutic treatment of fungal infections in animals, especiallymammals including humans, in particular humans and domesticated animals(including farm animals). The compounds may be used for the treatment oftopical fungal infections in man caused by, among other organisms,species of Candida (eg Candida Albicans), Trichophyton (eg Trichophytonmentagrophytes), Microsporum (eg Microsporum gypseum) or Epidermophytonor in mucosal infections caused by Candida Albicans (e.g. thrush andvaginal candidiasis). They may also be used in the treatment of systemicfungal infectious caused by, for example Candida albicans, Cryptococcusneoformans, Aspergillus fumigatus, Coccidiodes, Paracocciciodes,Histoplasma or Blasmtomyces spp. They may also be of use in treatingeumycotic mycetoma, chromoblastomycosis and phycomycesis.

42-O-demethylrapamycin and its pharmaceutically acceptable derivativesare also active as an immunomodulatory agents. The term"immunomodulatory agent" as used herein means that the compound of theinvention is capable of inducing immune suppression by inhibiting T (andB) cell responses in vitro and/or by producing a statisticallysignificant decrease in the intimation system response medicatedsecondary lesion in the adjuvant induced arthritis. Indications fortherapy include, but are not limited to, the treatment of the followingdisease states: rheumatoid arthritis, systemic lupus erythematosis,multiple sclerosis, acute transplantation/graft rejection, myastheniagravis, progressive systemic sclerosis, multiple myeloma, atopicdermatitis, hyperimmunoglobulin E, hepatitis B antigen negative chronicactive hepatitis, Hashimoto's thyroiditis, Familial Mediterranean fever,Grave's disease, autoimmune hemolytic anemia, primary biliary cirrhosis,inflammatory bowel disease, insulin dependent diabetes mellitus.

42-O-demethylrapamycin and its pharmaceutically acceptable derivativesshould also have activity against carcinogenic tumours. Morespecifically, the compounds should be useful for reducing tumour size,inhibiting tumour grown and/or prolonging the survival time ofturnout-bearing animals.

Accordingly the invention provides 42-O-demethylrapamycin or derivativesthereof for use in medical therapy, in particular for use as anantifungal agent or immunomodulatory agent, or as an agent againstcarcinogenic tumours.

The invention further provides a method of treating a human or animalsuffering from a fungal infection by the administration of an effectiveamount of 42-O-demethylrapamycin or derivative thereof.

Moreover, the invention provides a method of treating a human or animalin need of immunomodulation by administration of an effective mount of42-O-demethylrapamycin or derivative thereof.

The invention also provides a method of treating carcinogenic tumours ina human or animal comprising administering to such human or animal aneffective, non-toxic amount of 42-O-demethylrapamycin or derivativethereof.

The invention further provides a pharmaceutical composition comprising acompound of the formula (I) or a pharmaceutically acceptable saltthereof together with a pharmaceutically acceptable diluent or carrier.The composition is preferably for human use in tablet, capsule,injectable or cream form.

For human use 42-O-demethylrapamycin or derivatives thereof can beadministered alone, but will generally be administered in admixture witha pharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. For example, theymay be administered orally in the form of a tablet containing suchexcipients as starch or lactose, or in a capsule or ovule either aloneor in admixture with excipients, or in the form of an elixir orsuspension containing a flavouring or colouring agent. They may beinjected parenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, they are best used in theform of a sterile solutions which may contain other substances, forexample, enough salts or glucose to make the solution isotonic.

For oral and parenteral administration to human patients suffering froma fungal infection, it is expected that the daily dosage level of theantifungal compounds of formula (I) will be from 0.05 to 100 preferably0.1 to 10 mg/kg (in divided doses) when administered by either the oralor parenteral route. Thus tablets or capsules of the compounds can beexpected to contain from 5 mg to 0.5 g of active compound foradministration singly or two or more at a time as appropriate. Thephysician in any event will determine the actual dosage which will bemost suitable for an individual patient and will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Equally for a human patient in need of immunomodulation the dailyparenteral or oral dosage regimen for the compound or derivative thereofwill preferably be from 0.1 mg/kg to 30 mg/kg.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of compound or derivativethereof would be for the purpose of treating carcinogenic tumours.Generally, however, an effective dosage is expected to be in the rangeof about 0.05 to 100 milligrams per kilogram body weight per day.

No unacceptable toxicological effects are expected when the compound isadministered in the above mentioned dosage ranges.

The compounds and compositions according to the invention may beformulated for administration in any convenient way for use in human orveterinary medicine, by analogy with other antifungal, anticancer orimmunomodulatory agent.

The compounds and tablets and capsules for oral administration may be inunit dosage form, and may contain conventional excipients including, forexample, binding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrollidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; and pharmaceuticallyacceptable wetting agents, for example sodium lauryl sulphate. Thetablets may be coated according to methods well known in normalpharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives, including, for example, suspending agents, forexample sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel orhydrogenated edible fats; emulsifying agents, for example lecithin,sorbitan monooleate or acacia; non-aqueous vehicles (which may includeedible oils), for example almond oil, oily esters (for exampleglycerine), propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid; and, ifdesired, conventional flavouring and colour agents.

Compositions according to the invention intended for topicaladministration may, for example, be in the form of ointments, creams,lotions, eye ointments, eye drops, ear drops, impregnated dressings, andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

Compositions according to the invention may be formulated assuppositories, which may contain conventional suppository bases, forexample cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteraladministration may conveniently be in fluid unit dosage forms, which maybe prepared utilizing the compound and a sterile vehicle,propyleneglycol. The compound, depending on the vehicle andconcentration used, may be either suspended or dissolved in the vehicle.Parenteral suspensions may be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilisation cannot be accomplished by filtration. Thecompound may instead be sterilised by exposure to ethylene oxide beforebeing suspended in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in such suspensions in order to facilitateuniform distribution of the compound.

Compositions according to the invention may also be administered byinhalation. By "inhalation" is meant intranasal and oral inhalationadministration. Appropriate dosage forms for such administration such asan aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques.

The following examples serve to illustrate the present invention.

EXAMPLE 1

Preparation of 42-O-demethylrapamycin

A culture capable of producing 42-O-demethylrapamycin from rapamycin hasbeen classified as Streptomyces sp. and has been deposited in theNational Collection of Industrial and Marine Bacteria, 23, St. MacharDrive, Aberdeen AB2 1RY, Scotland, UK. under the accession number NCIMB40515.

Cultivation

Each of five 500 ml flasks containing 100 ml M2 medium arkasoy, 10 g/l;glycerol, 20 g/l; CoCl₂.6 H₂ O, 0.005 g/l; MgCl₂.2H₂ O, 0.1 g/l; FeCl₃,0.03 g/l; ZnCl₂, 0.005 g/l; CuCl₂.2H₂ O, 0.005 g/l; MnSO₄.4H₂ O, 0.005g/l pH 6.6 unadjusted! were inoculated with 2 plugs of agar from wellgrowing cultures on A3 agar yeast extract, 5 g/l; malt extract, 10 g/l;glycerol, 10 g/l; peptone soya, 5 g/l; agar No.3, 20 g/l; pH 6.5! inpetri dishes. Alternatively a large loopful of well growing culture froma petri dish was mixed with 3 ml of Tween 80 and the total contentsadded to 100 ml M2 media in a 500 ml flask, grown at 28° C., 240 rpm for3 days, 1 ml of the culture broth is then used to inoculate 100ml M2media in each of the five 500 ml flasks. The flasks were then grown at28° C. and 240 rpm.

Incubation

After 5 days, rapamycin (20 mg) as a solution in acetone (3.5 ml) wasadded to each flask. Rapamycin can be obtained from arapamycin-producing culture NCIMB 40319 deposited on 14 Sep. 1990, or byculturing a rapamycin producing organism eg NRRL 5491 as disclosed inU.S. Pat. No. 3,929,992 issued 30 Dec. 1975, the entire disclosure ofwhich is hereby incoporated by reference. Flasks were incubated at 28°C., 240 rpm for about 20 h.

Isolation procedure

Solvent extraction

The contents of the flasks was bulked and adjusted to pH4 with dilutesulphuric add. 500 ml of dichloromethane was added and the mixturestirred for 2 h, the organic solvent phase was recovered separating thephases by centrifugation and a further 200 ml of dichloromethane addedand stirred for 1 h. The organic solvent phases were combined andconcentrated in vacuo to an oil. To the oil was added 100 ml methanol,the methanol extract filtered and the filtrate concentrated to an oil invacuo.

Silica chromatography

The oil was loaded on a Kieselgel 60 (70-230 mesh) column (25×50 mm)packed in acetone:hexane (15:85). After loading, the column was elutedwith a step gradient of acetone-hexane. Fractions eluted after 35:65acetone:hexane contain 42-O-demethylrapamycin. These were combined andreduced to dryness in vacuo and stored at -20° C.

Preparative hplc

The stored solid was dissolved in 500 μl methanol and 100 μl portionsinjected separately onto a reverse phase Microsorb C-18 column and precolumn (21.4 mm×25 cm and 21.4×5 cm) (Rainin Instruments USA). Afterinjection elution continued with 78:22 methanol:H₂ O, at 6 ml/minute andwas monitored for UV absorbance at 278 nm. Fractions containing theobject compound from a total of 5 injections were pooled, concentratedin vacuo to remove the methanol and freeze dried.

Fractions containing the object compound were analysed by reverse phasehlpc using a Spherisorb S10ODS2 (PhaseSep) column (25 cm×4.6 mm) and aWaters pre-column. The column was monitored by UV absorption at 278 nmand eluted with 78:22 methanol-water at 2 ml/minute. Under theseconditions the object compound had a retention time of 6.8 minutes(differing from rapamycin, retention time of 8.4 minutes).

Spectroscopic Data

The resulting compound was characterised by mass spectroscopy (FAB) M+Na)⁺ =922 and by proton and ¹³ C nuclear magnetic resonancespectroscopy (see Table 1 below). UV spectroscopy shows UVl_(max) inacetone at 275 nm.

                  TABLE 1                                                         ______________________________________                                        Spectroscopic Data for 42-O-Demethylrapamycin:                                MS (FAB/NaCl) m/z 922 (M + Na).sup.+                                          1H-NMR (CDCl3, 400 MHz, 4:1 mixture of trans: cis amide rotamers;             data for the trans-rotamer):                                                  ppm       assign     muitipl    J's (Hz)                                      ______________________________________                                        6.383     H-4        dd         14.7, 10.5                                    6.310     H-3        dd         14.7, 9.8                                     6.134     H-2        dd         15.0, 9.8                                     5.970     H-5        d          1o.5                                          5.518     H-1        dd         15.0, 9.0                                     5.407     H-26       d          10.3                                          5.273     H-20       d          5.0                                           5.152     H-22       q          5.8                                           4.618     OH-13      s          --                                            4.197     H-28       br d       5.0                                           3.89      H-9        m          --                                            3.782     H-29       d          5.3                                           3.647     H-7        t          7.3                                           3.515     H-16       br d       11.9                                          3.338     MeO-29     s          --                                            3.4-3.25 overlapping multiplet of H-25, H-42 and H-43                         3.134     MeO-7      s                                                        2.710     H-23       dd         16.8, 5.8                                     2.587     H-23       dd         16.8, 5.7                                     1.758     Me-27      s          --                                            1.655     Me-6       s          --                                            1.085     Me-25      d          6.7                                           1.051     Me-33      d          6.4                                           0.985     Me-31      d          6.5                                           0.942     Me-12      d          6.6                                           0.905     Me-38      d          6.7                                           ______________________________________                                    

EXAMPLE 2 Bioactivity of 42-O-demethylrapamycin

The compound was analysed for antifungal and immunosuppressive activityusing the following bioassays:

A. Assay for antifungal activity

Yeast organism (Saccharomyces cerevisiae) in logarithmic growth wereplated on complete agar medium (YPD). Compounds dissolved in anappropriate aqueous or organic solvent were placed in wells punched inthe agar. Plates were incubated for 48 hours and zones of inhibitionwere measured. The potency of compounds were quantified by regressionanalysis of plots of inhibition zone versus the log of drugconcentration.

B. Mitogenesis Assay for Immunosuppresive Activity

Spleen cells for BDF1 female mice were established in RPMI with 10%fetal calf serum at 5×10⁶ /mL. One hundred mL aliquots of thissuspension (5×10⁵ cells) were dispensed into 96-well round-bottomedmicrotiter plates (Linbro, Flow Laboratories). Concanavalin A (5 μg/ml)was added as the mitogenic stimulus, and the final volume in themicrotiter wells was adjusted to 200 μL with RPMI. Cell cultures wereincubated for 72 hours at 37° C. in a 5% CO₂ atmosphere and pulsed with0.5 μCi ³ H-thymidine (specific activity 2.00 Ci/mole) for the last 18hours of the 72 hours culture. The cells were harvested on an automatedmultiple sample harvester and cell-associated radioactivity counted in aBeckman liquid scintillation counter. The results were expressed as themean values derived from quadruplicate measurements. Cell viability wasdetermined by trypan blue exclusion after 72 hours of incubation.Compounds to be tested were added to the microtiter plates at theappropriate dilutions prior to the addition of cells.

Results of these two assays for compounds of this invention are providedin Table 2.

                  TABLE 2                                                         ______________________________________                                                          a                                                                             Antifungal                                                                             b                                                  Compound          Activity Mitogenesis                                        ______________________________________                                        Control           0.0061   1 ± 1                                           (rapamycin)                                                                   42-O-demethylrapamycin                                                                          0.012    3                                                  0.013             --                                                          ______________________________________                                         a Assay: (IC.sub.12, μg/ml)                                                b Assay: (IC.sub.50, nM)                                                 

EXAMPLE 3 COMPOSITION EXAMPLES A--H

A--CAPSULE COMPOSITION

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg of a compound of the invention, in powdered form, 100 mg of lactose,32 mg of talc and 8 mg of magnesium stearate.

B--INJECTABLE PARENTERAL COMPOSITION

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5% by weight of acompound of the invention in 10% by volume propylene glycol and water.The solution is sterilized by filtration.

C--OINTMENT COMPOSITION

Compound of the invention 1.0 g

White soft paraffin to 100.0 g

The compound of the invention is dispersed in a small volume of thevehicle and granually incorporated into the bulk of the vehicle ofproduce a smooth, homogeneous product. Collapsible metal tubes are thenfilled with the dispersion.

D--TOPICAL CREAM COMPOSITION

Compound of the invention 1.0 g

Polawax GP 200 20.0 g

Lanolin Anhydrous 2.0 g

White Beeswax 2.5 g

Methyl hydroxybenzoate 0.1 g

Distilled Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60° C. Thecompound of the invention is then added and dispersed throughout, andthe composition is allowed to cool with slow speed stirring.

E--TOPICAL LOTION COMPOSITION

Compound of the invention 1.0 g

Sorbitn Monolaurate 0.6 g

Polysorbate 20 0.6 g

Cetostearyl Alcohol 1.2 g

Glycerin 6.0 g

Methyl Hydroxybenzoate 0.2 g

Purified Water B.P. to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of thewater at 75. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75 C and added to the aqueous solution.The resulting emultion is homogenized, allowed to cool with continuousstirring and the compound of the invention is added as a suspension inthe remaining water. The whole suspension is stirred until homogenized.

F--EYE DROP COMPOSITION

Compound of the invention 0.5 g

Methyl Hydroxybenzoate 0.01 g

Propyl Hydrobenzoate 0.04 g

Purified water B.P. to 100.00 ml (B.P.=British Pharmacopia) The methyland propyl hydroxybenzoates are dissolved in 70 ml purified water at 75°C. and the resulting solution is allowed to cool. The compound of theinvention is then added, and the solution is sterilized by filtrationthrough a membrane filter (0.22 mu m pore size) and packed asepticallyinto suitable sterile containers.

G--COMPOSITION FOR ADMINISTRATION BY INHALATION

For an aerosol container by a capacity of 15-20 ml: Mix 10 mg of acompound of the invention with 0.2-0.2% of a lubricating agent, such aspolysorbate 85 or oleic acid, and disperse such mixture in a propellant,such as freon, preferably in a comination of (1,2dichlorotetrafluoroethane) and difluorochloromethane and put into anappropriate aerosol container adapter for either tranasal or oralinhalation administration.

H--COMPOSITION FOR ADMINISTRATION BY INHALATION

For an aerosol container with a capacity of 15-20 ml; Dissolve 10 mg ofa compound of the invention in ethanol (6-8 ml), add 0.1-0.2% of alubricating agent, such as polysorbate 85 or oleic acid; and dispersesuch in a propellant, such as freon, preferably a combination of (1.2dichlorotetrafluoroethane) and difluorochloromethane, and put into anappropriate aerosol container adapter for either intranasal or oralinhalation administration.

I claim:
 1. A process for the production of a compound of formula (I):##STR3## which is 42-O-demethylrapamycin, or pharmaceutically acceptablesalt thereof, which comprises contacting a microorganism which belongsto the genus Streptomyces with rapamycin and subsequently isolating said42-O-demethylrapamycin, or a pharmaceutically acceptable salt thereof,from the incubation.
 2. A process according to claim 1, which comprisesseparating said compound or a pharmaceutically acceptable salt thereof,from a solution thereof in admixture with other antibacterially activesubstances and/or inactive substance by adsorption onto an adsorbentresin.
 3. A process as claimed in claim 1, wherein the producingmicroorganism is Streptomyces NCIMB
 40515. 4. Streptomyces NCIMB 40515.